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Raman micro-spectroscopy and different approaches for multivariate analysis were used for an investigation of subcellular regions of X-ray exposed single SH-SY5Y human neuroblastoma cells. Nucleus and cytoplasm regions of single cells were investigated after X-rays irradiation (0, 2, 4, 6 and 8 Gy). Cells fixed immediately after irradiation and 24h irradiation were considered. Principal component analysis (PCA) and interval-PCA (i-PCA) were used for analyzing the spectra in order to highlight the changes due to the different treatments. Biochemical changes occurring in the nucleus and cytoplasm regions of single cells upon X-ray irradiation were observed. The analysis of Raman spectra allowed us to detect modifications in the contribution from proteins, nucleic acids, lipids, and carbohydrates of cells, induced at different extent on the two cell regions. The biochemical changes occurring in these cells were also discussed by using an alternative approach, namely the analysis of difference spectra, obtained by subtracting the cytoplasm-related spectrum from the corresponding one detected at the nucleus. The proposed approach enabled us to evidence some features not outlined in previous investigations. The results showed that it is possible to study in a selective way the effects of ionizing radiation on different neuroblastoma cell spatial regions. An increase of the signal related to the nucleobases, a decrease of DNA and/or RNA backbone contribution, a protein rearrangement with changes in the secondary structure, and an increase in lipid saturation were observed. These results indicated that the development of accurate data analysis methods enabling to take into account the complexity of the Raman spectra of cells and tissues and the high number of spectra needed to consider the intrinsic variability of biological samples.
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